Coordinate Regulation of Stress- and Mitogen-Activated Protein Kinases in the Apoptotic Actions of Ceramide and Sphingosine

  1. W. David Jarvis1,
  2. Frank A. Fornari, Jr.2,
  3. Kelly L. Auer3,
  4. Alex J. Freemerman1,
  5. Eva Szabo6,
  6. Michael J. Birrer6,
  7. Charlene R. Johnson4,
  8. Suzanne E. Barbour4,
  9. Paul Dent3,5 and
  10. Steven Grant1,4,5
  1. Departments of 1Medicine (W.D.J., A.J.F., S.G.), 2Medicinal Chemistry (F.A.F.), 3Radiation Oncology (K.L.A., P.D.), 4Microbiology/Immunology (C.R.J., S.E.B., S.G.), and 5Pharmacology/Toxicology (P.D., S.G.), Medical College of Virginia, Richmond, Virginia 23298, and 6Biomarkers and Prevention Research Branch (E.S., M.J.B.), National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20850

    Abstract

    We characterized participation of the stress-activated protein kinase (SAPK) cascade in the lethal actions of the cytotoxic lipid messengers ceramide and sphingosine in U937 human monoblastic leukemia cells. Acute exposure of U937 cells to either lipid resulted in loss of proliferative capacity, degradation of genomic DNA, and manifestation of apoptotic cytoarchitecture. Ceramide robustly stimulated p46-JNK1/p54-JNK2 activity and increased expression of c-jun mRNA and c-Jun protein; in contrast, sphingosine moderately stimulated p46-JNK1/p54-JNK2 and failed to modify c-jun/c-Jun expression. Dominant-negative blockade of normal c-Jun activity by transfection with the TAM-67 c-Jun NH2-terminal deletion mutant abolished the lethal actions of ceramide but was without effect on those of sphingosine, indicating that ceramide-related apoptosis is directly dependent on activation of c-Jun, whereas sphingosine-induced cell death proceeds via an unrelated downstream mechanism. Characterization of the mitogen-activated protein kinase (MAPK) cascade in these responses revealed a further functional disparity between the two lipids: basal p42-ERK1/p44-ERK2 activity was gradually reduced by ceramide but immediately and completely suppressed by sphingosine. Moreover, blockade of the MAPK cascade by the aminomethoxyflavone MEK1 inhibitor PD-98059 unexpectedly activated p46-JNK1/p54-JNK2 and induced apoptosis in a manner qualitatively resembling that of sphingosine. Both lipids sharply increased p38-RK activity; selective pharmacological inhibition of p38-RK by the pyridinyl imidazole SB-203580 failed to mitigate the cytotoxicity associated with either ceramide or sphingosine, suggesting that p38-RK is not essential for lipid-induced apoptosis. These findings demonstrate that reciprocal alterations in the SAPK and MAPK cascades are associated with the apoptotic influence of either lipid inasmuch as (i) ceramide-mediated lethality is primarily associated with strong stimulation of SAPK and weak inhibition of MAPK, whereas (ii) sphingosine-mediated lethality is primarily associated with weak stimulation of SAPK and strong inhibition of MAPK. We therefore propose that leukemic cell survival depends on the maintenance of an imbalance of the outputs from the MAPK and SAPK systems such that the dominant basal influence of the MAPK cascade allows sustained proliferation, whereas acute redirection of this balance toward the SAPK cascade initiates apoptotic cell death.

    Footnotes

    • Send reprint requests to: Dr. W. David Jarvis, Medical College of Virginia, MED-HEM/ONC, Box 980230, MCV Station, Richmond, VA 23298. E-mail: wjarvis{at}gems.vcu.edu

    • Portions of this work were presented in preliminary form at the Keystone Symposium on Cell Biology entitled Apoptosis (Programmed Cell Death), Tamarron, CO, March 5–11, 1995, and the 87th Annual Meeting of the American Association for Cancer Research, Washington, D.C., April 20–24, 1996.

    • This work was supported primarily by National Cancer Institute Research Grant CA63753 and Leukemia Society of America Award 6405-97 (S.G.). Other support includes National Cancer Institute National Research Service Award CA09380 (W.D.J.), National Heart, Lung, and Blood Institute National Research Service Award HL09241 (F.A.F.), United States Public Health Service Training Grants CA09564 and DK07150 (A.J.F., K.L.A.), and National Cancer Institute Research Grant IN-105V (P.D.). Additional funding was provided by the A. D. Williams Foundation of the Medical College of Virginia; the Robert B. Dalton Endowment Fund and the Thomas F. and Kate Miller Jeffres Memorial Trusts; and by National Cancer Institute Cancer Center Support Core Grant CA-16059 to the Massey Cancer Center.

    • Abbreviations:
      SAPK
      stress-activated protein kinase
      KSR
      kinase suppressor of ras
      CAPK
      ceramide-activated protein kinase
      CAPP
      ceramide-activated protein phosphatase
      ERK
      extracellular signal receptor-activated kinase
      JNK
      c-Jun NH2-terminal kinase
      MAPK
      mitogen-activated protein kinase
      PKC
      protein kinase C
      SMase
      sphingomyelinase
      TAM-67
      c-jun/c-Jun transactivation-deficient mutant
      AP
      activator protein
      cPKC
      group A (conventional) isoform of protein kinase C
      nPKC
      group B (novel) isoform of protein kinase C
      PBS
      phosphate-buffered saline
      TdT
      terminal deoxynucleotidyl transferase
      EGTA
      ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid
      HEPES
      4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
      MAPP
      N-myristoylamino-1-phenyl-1-propanol
      bp
      base pair(s)
      PLA
      phospholipase A
      FITC
      fluorescein isothiocyanate
      TBST
      Tris-buffered saline containing 1% Tween 20
      • Received May 7, 1997.
      • Accepted August 14, 1997.
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    1. Molecular Pharmacology December 1, 1997 vol. 52 no. 6 935-947
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